CN1223660A - Method and compounds for curing diseases caused by mutations - Google Patents

Abstract

The invention concerns the use of duplex oligonucleotides having both 2'-deoxyribonucleotides and ribonucleotides, wherein there is base pairing between the two types of nucleotides. The sequence of the oligonucleotide is selected so that the 3' and 5' most regions of the oligonucleotides are homologous with (identical to) the sequence of a preselected target gene of a cell. The two regions of homology embrace a region that is heterologous with the target sequence. The introduction of the oligonucleotide into the nucleus of the cell causes the alteration of the target gene such that the sequence of the altered target gene is the sequence of the heterologous region. Consequently, the oligonucleotides of the invention are termed chimeric mutation vectors (CMV). In one embodiment of the invention the target gene is a globin gene and the target cell is a hematopoietic stem cell. This embodiment can be used to correct certain hemoglobinopathies such as Sickle Cell Disease, beta-thalassemia and Gaucher Disease. The rate of correction of the globin gene is high enough so that no selection of the treated hematopoietic stem cells is required to obtain a therapeutically significant effect. In one embodiment the ribonucleotides of the CMV contain methylated 2'-O.

Description

The method and the compound of the disease that the treatment sudden change causes

I. invention field

The field of the invention relates to the treatment of diseases that sudden change causes, described sudden change causes that hematopoietic cell maybe can be from the abnormal level or the unusual product of the mutator gene the taking-up of curee's human body, cultivation and other cell type of implanting again.Homologous recombination by mutator gene and chimeric repair vector (CRV) makes the pairing of patient's mutator gene realize treatment, and CRV is the nucleic acid that existing deoxyribonucleotide has ribonucleotide again.More particularly, this field relates to the reparation of the sudden change that causes sickle cell disease, β-thalassemia and familial splenic anemia.

II. the disease that the sudden change of background of invention A. hematopoietic cell expressing gene causes and with the treatment of bone marrow transplantation

1. hemoglobinopathy and familial splenic anemia

The known structure variation body that more than 500 kind of oxyphorase arranged.There are many people of variation oxyphorase to be asymptomatic or to have only slight influence.Three common varients are just relevant with important disease.HbS (the sicklemia red eggs are white) and two kinds of oxyphorases of HbC are in the codon of coding beta-globin Glu6 point mutation to have taken place, and these two kinds of globins have discovery in African and descendant thereof.HbC is first displacement that G → A takes place owing to the 6th codon, and HbS then is because A → T displacement has taken place second of the 6th codon, has produced Lys respectively ⁶And Val ⁶The varient HbE that the third of the oxyphorase relevant with disease is common is because coding Glu ²⁶The first location of the 26th codon the displacement of G → A has taken place, the result has produced Lys ²⁶, HbE finds in South East Asia crowd and descendant thereof at first.

The people of HbS, HbC or HbE heterozygosis does not have tangible illness.Yet the allelic heterozygote of HbS homozygote, HbS/HbC heterozygote and HbC or HbE and β-thalassemia just has a strong impact on, and needs frequent Medicare.

Thalassemia is a class hemoglobinopathy, and wherein α-Zhu Danbai and beta-globin resultant velocity are unbalanced.Thalassemia can be divided into α or β type according to mutator gene, and is to reduce fully or partial reduction is divided into for example α according to influenced albumen ⁰And β ⁰(reduction) fully or as α ⁺And β ⁺(partial reduction).

Two function α-Zhu Danbai genes that closely link to each other are arranged in the monoploid human genome.The most general reason of α-Di Zhonghaipinxue disease is that disappearance or rearrangement have taken place for the α-Zhu Danbai gene or the α-Zhu Danbai region that link to each other.

On the contrary, the common reason of β-thalassemia is point mutation.Common reason is that G → A displacement takes place 0 of beta-globin gene 11, new montage acceptor site of generation in first intron (IVSI).

Characterization about 100 kinds of dissimilar β-thalassemias.Described in the sudden change of characteristic and had only 14 kinds to be dominance, caused the significant clinical patient's condition during promptly with wild-type beta-globin allelotrope heterozygosis.Remaining causes the serious clinical patient's condition when suddenling change with second β-thalassemia gene pure or heterozygosis, is called the major thalaseemia disease.The detailed summary of hemoglobinopathy is seen outstanding the 7th edition the 113rd chapter of " metabolism of inherited disease and molecular basis " (THE METABOLIC AND MOLECULAR BASIS OF INHERITEDDESEASE) (McGraw-Hill such as Weatherall, New York, 1995).

Familial splenic anemia is the lysosome glycolipidosis.Three kinds of generally acknowledged types are arranged.1 type familial splenic anemia is the most general in the Jew of Ashkhabad (Eastern Europe) and descendant thereof; 2 types are panethinc; 3 types are the most general in Sweden the north.The cause of disease of familial splenic anemia is a glucose cerebrosidase defectiveness, and this enzyme also is acid Polyglucosidase.Glucocerebrosidase is expressed in the cell type of the scavenger cell/monocyte that comes from marrow.

The displacement of the 1225th Nucleotide generation A → G of the three kinds of different common mutations that causes familial splenic anemia: mRNA is arranged, cause the 370th residue of glucocerebrosidase that the displacement (N370S sudden change) of Asn → Ser takes place; T → C displacement takes place in the 1448th Nucleotide of mRNA, causes glucocerebrosidase the 444th residue that Leu → Pro takes place and replaces (L444P sudden change); And the Nucleotide G repeat to cause phase shift mutation (84GG sudden change).N370S is relevant with the Ashkhabad crowd with the 84GG sudden change, and L444P suddenlys change relevant with the northern crowd of Sweden and is sporadic sudden change.

The uncommon but unrare sudden change that causes the encoding sequence of familial splenic anemia has been identified as follows: in 754 T → C; In 1192, C → T; In 1193, G → A; In 1297, G → T; In 1342, G → C; In 1504, C → T; And in 1604, G → A.

The sudden change that causes familial splenic anemia is recessive, promptly has an allelic individuality of wild-type glucocerebrosidase not have symptom.N370S sudden change is isozygotied individually develops into slight disease late period at its life usually.On the contrary, the homozygous individual of L444P sudden change suffers from 2 more serious types and 3 type familial splenic anemia.The 84GG sudden change causes product not have enzymic activity when homozygotic state, and causes serious clinical disease.In uncommon sudden change, in the sudden change of Nucleotide 754,1192,1297 and 1342 and the disease-related of severe form.The detailed summary of familial splenic anemia is seen Beutler, E. and Grabowski " metabolism of genetic diseases and molecular basis " (THEMETABOLIC AND MOLECULAR BASIS OF INHERITED DESEASE) the 7th edition, the 86th chapter (McGraw-Hill, New York, 1995).

2. treat the disease that sudden change causes by bone marrow transplantation

Serious β-thalassemic the case of allogeneic bone marrow transplantation treatment of the marrow with normal beta-globin gene by HLA coupling has been reported clinical benefit.Giardini, C etc., 1995, Annu.Rve.Med.46:319-30 (thalassemia); Lucarelli, G etc., 1991, Hematol.Oncoi.Clin.North Am.5:549 (thalassemia); Kalinyak, K.A etc. 1995, Am.J.of Hemat.48:256-61 (sickleshaped cell); Abboud, M.R. etc. 1994, Am.J.ofPed.Hem/Onc 16:86-89 (sickleshaped cell); Kirkpatrick, D.V etc. 1991, Semi.Hematol.28:240 (sickleshaped cell).Clinical effectiveness shows that when moving into successfully be medicable.Immigration can be persistent; Have and do not find repulsive interaction among the 3-8 that is reported in subsequently.Yet because the development of graft versus host disease (GVH disease) and as the maintenance dose of the immunosuppressive drug of S-Neoral, the ratio of failure is tangible.The marrow that obtains the HLA coupling also has suitable difficulty.

The allogeneic bone marrow transplantation for the treatment of 1 type familial splenic anemia has produced the result roughly the same with above-mentioned thalassemia.Ringden, O. etc. 1995, Transplantation 59:864); Chan, K.W. etc. 1994, Bone Marrow Transplantation 14:327.Bone marrow transplantation is used for the treatment of more serious 2 types that relate to central nervous system and 3 type familial splenic anemia and it is reported and also obtained satisfied result.Tsai, P. etc. 1992, Pediatr.Res.31:503; Svennerholm, L. etc. 1991, Dev.Neurosci.13:345-51.Compare with globin gene, can obtain the glucocerebroside expression of enzymes of level of significance with virus expression carrier, this just autotransplantation of prompting transduction marrow be effective therapy to familial splenic anemia.Karlsson, S. and Correll, P.H., 1993, Bone Marrow Transplantation 11 (supplementary issue 11): 124-7.B. have a chimeric oligonucleotide of dna rna base pair

Have complementary DNA Nucleotide and ribonucleotide and contain oligonucleotide with the fragment homologous sequence of phage M13mp19, among the Mol.and Cell.Biol.14:7163-7172 description is arranged in November, 1994 such as Kmiec.E..This oligonucleotide has single adjacent Nucleotide section.Kmies etc. point out that this oligonucleotide is the substrate that comes from the REC2 autosyndetic pairing enzyme of Ustilago maydis.

That announce June 15 nineteen ninety-five and be the patent specification WO 95/15972 of the u.s. patent application serial number 08/353,657 submitted on December 9th, 1994 accordingly, the chimeric repair vector (CRV) of introduction hereditary change in prokaryotic cell prokaryocyte has been described.Existing example report in Ustilago maydis gene and mouse ras gene.Back one example design is used for being incorporated into the ras gene transforming sudden change, makes the successful sudden change of ras gene in NIH 373 cells can make cell colony growth (" conversion ").WO 95/15927 specification sheets has been reported the maximum conversion rate of NIH 373 less than 0.1%, promptly per 10 ⁶About 100 transformants of individual cell are subjected to the effect of ras CRV.The transformant frequency of occurrences approximately is 600/10 in the Ustilago maydis system ⁶At Kmiec, E.B., has description among the Seminar inOncology 23:188 in February, 1996 the chimeric vector of sudden change introducing people bcl-2 gene in design.

Design is repaired the CRV of sudden change in the codon 12 of K-ras at Kmiec, E.B February nineteen ninety-five, among the Advanced Drug Delivery Reviews 17:333-40 description is arranged.With the lipofection body of laws CRV is introduced the Capan2 cell, in the clone Capan 2 that is derived from human pancreas cancer, detect CRV.CRV is after 24 hours in contact, and harvested cell also extracts genomic dna; The fragment pcr amplification that contains K-ras codon 12, and by estimating transformation efficiency with allelic specific probe hybridization.The report transformation efficiency is near 18%.

Design is repaired the CRV of coding liver/bone/kidney type alkaline phosphatase gene sudden change at Yoon, and K. etc., have report among the Proc.Natl.Acad.Sci 93:2071 in March, 1996.Described alkaline phosphatase gene is with the instantaneous introducing Chinese hamster ovary celI of plasmid, introduce CRV after 6 hours.The CRV introducing is reclaimed plasmid and is analyzed after 24 hours.The result shows the alkaline phosphatase gene of repairing nearly 30-38% with CRV.

III. summary of the invention

The invention provides the method for the human subject inherited disease that causes of treatment mutant target gene.The present invention has comprised the reparation of the pathogenic mutation of curee's cell type gene, wherein can take out cell type from the curee, cultivate and by carrying out external reparation in the cell that CRV is incorporated into cell type, and then implant in curee's body, the sudden change reparation of wherein replanting into cell or its filial generation is medicable.In general, the reparation of the dominant mutation more than the reparation of the recessive mutation of cell colony more than 25% of cell type and 50% is that the effective for repairing rate is gone up in treatment.

In one embodiment of the invention, target gene is the type that is referred to as normal expression among the offspring of the marrow myelocyte of " hematopoietic cell " or medullary cell hereinafter.Therefore, one embodiment of the invention are a kind of like this methods, comprise that the hemopoietic stem cell (HSC) the curee places substratum, chimeric repair vector (CRV) is introduced among the HSC repaired target mutation, again the HSC that comprises CRV is implanted into the step that waits in curee's body.

The present invention has comprised the reparation of any sudden change resetting in big section disappearance or insertion (being the disappearance or the insertion of about 6 above bases), transposition and karyomit(e).In optimum implementation, the present invention includes single base substitution (" point mutation ") and the disappearance of 1,2 or 3 adjacent base or the reparation of insertion.

Other embodiment of the present invention is CRV, and CRV is the oligonucleotide of about 40 to 100 Nucleotide, and wherein about 12 to 46 Nucleotide have 2 '-hydroxyl or 2 '-methylol.Described CRV comprises the section identical with the target gene wild-type sequence, and this section is crossed over the sudden change that causes curee's disease.This CRV is made up of a double chain oligonucleotide, and promptly except oligonucleotide is formed two districts of about 4 unpaired nucleotides of hair clip end, its nucleotide base is the Watson-Crick paired.In specific embodiments, the present invention includes the CRV of the sequence of the human glucocerebrosidase gene of tool and globin gene and human beta-globin promotor.

IV. the accompanying drawing summary

Fig. 1. the general type of chimeric repair vector embodiment.

Fig. 2 A and 2B. Fig. 2 A have shown sequence and the structure of oligonucleotide Dh1 (SEQ ID NO:91) and chimeric oligonucleotide Ch1 (SEQ ID NO:88), Ch2 (SEQ ID NO:89) and Ch3 (SEQID NO:90).Fig. 2 B has illustrated the sequence of CRV Ch1 and the relation between the alkaline phosphatase gene (SEQ ID NO:92).The Nucleotide of DNA is capitalization; The Nucleotide of RNA is small letter.

Fig. 3. β ^S-globin (the Nucleotide 1-25 of SEQ ID NO:93), β ^AThe sequence of the codon 3-9 of-globin (the Nucleotide 1-25 of SEQ ID NO:94), δ-globin (SEQ ID NO:98) and chimeric vector SC1-SC5 (SEQ ID NO:93-97) and the adjacent dinucleotides of codon 2 and 10.The Nucleotide of DNA and RNA is shown in Fig. 2 A.

Fig. 4 A and 4B. Fig. 4 A and 4B be presented at lymphoblast that EB transforms cultivate in respectively with nM SC1 and nM SC2 become by β ^SChange β into ^AThe function and the β of beta-globin copy ^AChange β into ^SThe function of beta-globin copy.

Fig. 5. Fig. 5 has shown with joining cd34 ⁺Ng SC2 in the hemopoietic stem cell and become by β ^A-β ^SThe function of beta-globin copy.

V. detailed Description Of The Invention

The invention provides chimeric repair vector (CRV) and its application process, the method is called " chimeric diorthosis (chimeroplasty) ", is used for proofreading and correct the detrimental mutation without the transformant type that can instantaneously place cell to cultivate from human subject. Chimeric integration art is such process: suitable cell colony (target cell) is placed cultivation, carry out or do not grow, again the cell of cultivating is exposed to CRV, then cell is transplanted in this curee's body again. The present invention is partly based on this discovery: use CRV according to the present invention and cause the target gene more than 30% to obtain repairing, and can cause the target gene more than 50% to obtain repairing.

In one embodiment, described target cell is hematopoietic cell, particularly candidate stem cell. Here the hematopoietic cell of usefulness comprises precursor and the mature cell of the pedigree of red blood cell class, lymph sample monocyte sample (macrophage) and granulocyte. Candidate stem cell used herein (HSC) be included in find in marrow or the peripheral blood and also can repopulate marrow district (bone marrow space) and produce hematopoietic lineage offspring's all cells. A. the present invention is treated responsive disease

The present invention can be used for treating the generation of the abasement gene product that sudden change causes in the hematopoietic cell or normal gene product is expressed excessive or the not enough genetic disease that causes of expression, and wherein said sudden change does not comprise in large section insertion or deletion mutation or the chromosome resets or transposition.

Large section is inserted or deletion mutation is to compare with normal or wild-type sequence the sudden change that surpasses 6 adjacent nucleotides insertions or disappearance is arranged. Can comprise with the mutation type of the present invention's treatment: nucleotides is comprised the displacement of 3 of as many as and 6 adjacent nucleotides of as many as by the sudden change that different nucleotide subsitutions cause; Any sudden change that the insertion of 3 adjacent nucleotides of as many as or disappearance cause; Or the sudden change that 6 adjacent nucleotides of as many as insert or disappearance causes. Can be called " CRV can repair sudden change " here with the mutation type of the present invention's treatment.

It can be any Human genome that CRV can repair sudden change.Here the gene of usefulness refers to or structure gene, as the exon and the intervening sequence of the gene of coded protein; Or controlling elements, as the promotor or the enhanser of structure gene.The present invention also comprises same intragenic several CRV can repair the treatment of diseases that sudden change causes.Single CRV can proofread and correct the sudden change of the non-adjacent points in about 30 Nucleotide each other.Perhaps, can repair sudden change, disease is obtained medical treatment so that repair each CRV with a CRV in the mixture by the mixture of a plurality of CRV being introduced curee's HSC.The present invention comprises the CRV sequence of the Human genome that can repair any such sudden change, the Human genome of described sudden change in hematopoietic cell or HSC filial generation expression or lack that to express or express excessive be this curee's the cause of disease.In one embodiment, the present invention has eliminated the ras gene, sees Taparowski, E.1982 Nature 300:762; Sukumar, S. etc., 1983, Nature 306:658; In selectable embodiment, the present invention has eliminated ras gene and alkaline phosphatase gene, Weiss, and M.J. etc. 1988, Proc.Natl.Acad.Sci.85:7666.

Can comprise with the non-limiting case of the present invention's treatment: CRV can repair the hemoglobinopathy that sudden change causes in the globin structure gene, as the sickle-cell anemia disease; β-thalassemia causes the insufficient disease of beta-globin genetic expression for CRV in beta-globin promotor or the beta-globin structure gene can repair sudden change; One or more CRV can repair the familial splenic anemia type that sudden change causes in glucocerebrosidase structure gene.Noun used herein " structure gene " refers to the DNA of encoding gene product; Noun " gene " comprises regulates sequence (being promotor and enhanser), intron and exon.Noun " gene order " refers to the coding strand sequence of gene.Therefore its complementary sequence is the noncoding strand sequence.

One or more CRV among any curee can repair the position of sudden change and identify with technology well known to those skilled in the art.The sudden change position of sickle-cell disease (SCD) is in the codon of the 6th codon of coding beta-globin.

Except responsible sudden change position is a few cases of knowing, need curee's target gene be checked order, so that determine the position of point mutation.More than 500 point mutation of globin gene for example described.(Bunn,H.F.&Forget.B.G.,1986,HEMOGLOBIN:MOLECULAR,GENETIC?AND?CLINICAL?ASPECTS,W.B.Saunders,Phil.)。Equally, none sudden change causing familial splenic anemia (Hong, C.M. etc., 1990 DNA CellBiol 9:233-41) or β-thalassemia (Kazazian, H.H., 1990, Seminars inHematology 27:209-228).Those skilled in the art know the technology of identifying the specified point sudden change.The recovery of B.HSC, cultivation and transfection

Technology with those skilled in the art know now or develop reclaims hemopoietic stem cell (HSC) from curee's peripheral blood or marrow.The technology of being familiar with by those skilled in the art easily obtains HSC from the curee with the method for single blood sampling composition art.In one embodiment, the curee was taken with granulocyte colony-stimulating factor and during four days of cell list blood sampling composition art in 3 days in advance continuously.The monocyte of single blood sampling composition art separates by density gradient centrifugation or suitable process, takes out the cell that adheres to, with the antibody separation HSC of anti-CD34.Be fit to implement the present invention with CellPro and commercially available pillar or their Equivalent of Isolex trade mark.

The sickle-cell disease patient is subject to can be by giving the influence with the sedimentary sickleshaped cell of G-CSF crisis (crisis).Therefore, before crisis (pre-apheresis crisis) beginning, the curee should give preventative exchange transfusion before single blood sampling composition art.

Can be used for any technology of DNA transfectional cell by known or exploitation now, use the CRV transfectional cell.These technology comprise that electroporation, liposome shift and calcium phosphate precipitation.In one embodiment, this transfection with liposome transfer compounds such as DOTAP (N-[1-(2,3-two oily acyloxy) propyl group]-N, N, N-TMA (TriMethylAmine) mesylate, Boehringer-Mannheim) or its Equivalent finish.The amount of CRV is not conclusive to enforcement of the present invention, uses 10nM/10 ⁵Individual cell just can obtain good result.Operable ratio is per 10 ⁵Individual cell contains the 3 μ g DOTAP of 500ng CRV.Can use the rotaring dyeing technology among the following embodiment 1-3, be improved at serum free medium, added in the substratum of human serum albumin or human serum and cultivated cells transfected.

In one embodiment of the invention, be exposed to immediately in the mixture of CRV and liposome transfer compounds after the HSC of single blood sampling composition art separates, made its transfection in the about 6-16 of incubation hour.Wash cells transfected then and remove unabsorbed liposome, the technology cryopreservation of knowing according to the bone marrow transplantation field.Cryopreservation is finished in containing the substratum of 10%DMSD, and rate of cooling approximately is 3 ℃/minute.C. the transplanting of the HSC of Xiu Fuing

Be used for carrying out in the patient of sickle-cell disease and β-thalassemia any technology of the bone marrow precursor allosome transfer techniques of HLA coupling now, the HSC that all can be used for repairing is implanted among the curee.After single blood sampling composition art obtained medullary cell, this curee stood the cell reduction process immediately.Totally need the irradiation of 800-1200Rads dosage.Perhaps use the medullary cell toxic agents.Can use the curee to prepare to accept the system that bone marrow transplantation is used.Can use such system: transplant the composition that used busulfan 3.5mg/Kg/ days and endoxan 50mg/Kg/ days in preceding four days.Perhaps,, just use the dosage of 4.0mg/Kg/ days busulfans to add on a small quantity or do not add endoxan again, to obtain unusual effect if host's marrow does not break away from fully.The HSC that repairs can pass through the peripheral vein infusion.The reparation CD34 of infusion ⁺The total dose of cell can be to rebuilding the effective any dosage of patient's marrow.The normally about 1-4 of effective dose * 10 ⁶Individual CD34 ⁺Cell (HSC)/Kg.

Behind the HSC infusion of repairing, vein was given and reorganization 10 μ g/k/ days granulocyte colony-stimulating factors in preceding 48 hours, reached 1.5 * 10 in continuous three days up to the neutrophilic granulocyte absolute number ⁹/ L.The bone marrow transplantation field of thalassemia and sickle-cell disease is described by following document: Giardini, C. etc., 1995, Ann.Rev.Medicine 46:319-30; Abboud, M.R., American.J.of Ped.Hematol./Oncol.16:86-89; Storb, R. etc., 1991, Seminars in Hematology 28:235-39.D. the structure of chimeric repair vector

Chimeric repair vector (CRV) is the nucleic acid of 3 ', 5 '-connection, one 3 ' terminal and one 5 ' end is arranged at the most, a nearly 40-100 Nucleotide.In selectable embodiment, 3 ' and 5 ' end can be covalently bound.3 ' when not being connected with 5 ' end, just says that CRV has otch.CRV contains unpaired Nucleotide, forms 1 or the turnover of 2 hair clips, and described turnover is divided into two chains to CRV, makes that at least 15 bases of article one chain and the base of second chain are the Watson-Crick paired.Another feature of CRV is the section that has many at least three adjacent bases, and these sections are made up of 2 '-O or 2 '-alkyl oxide ribonucleotide, and the deoxyribonucleotide of these ribonucleotides and second chain is the Watson-Crick paired.

Noun used herein " district " refers to the part of polynucleotide, and its sequence has some specific source, and for example CRV has the district of at least 15 Nucleotide to have the sequence of human beta-globin gene fragment.Section is the part that the CRV of structural meaning is arranged.Given section or given area comprise 2 '-deoxynucleotide and ribonucleotide.Yet " ribonucleotide section " or " 2 '-deoxyribonucleotide section " only contains ribonucleotide and 2 '-deoxyribonucleotide.

Fig. 1 has shown the structure of the CRV of an embodiment, and this CRV has section (a)-(h).The diagrammatic purpose is for the 3 ' terminal 3 ' end at section (a) of CRV is described, 5 ' end is presented at 5 ' end of section (h), and promptly otch is positioned at (a) and (h) boundary between the section.Yet, if otch is arranged, the position of otch and be not crucial with respect to the CRV 3 ' of described section and the orientation of 5 ' direction.Put on (a)-(h) in order described section.In the embodiment of Fig. 1, first district comprises section (c), (d) and (e), with second district that forms by (a) section be complementary.

In one embodiment, the length and the characteristic of described section are as follows: section (a) is a 16-40 Nucleotide, preferably 20-30 Nucleotide.The sequence in section (a) district can be normal (the being wild-type) sequence of allelotrope coding strand or the sequence of noncoding strand that comprises the mutation gene (target gene) that will repair.Here the statement of usefulness: a district has the specific gene sequence fragments sequence, the meaning is that it has the coding strand sequence that comes from this gene, when the fragment sequence of the sequence in a section or district and coding strand or noncoding strand is identical, just say that maybe this district and described gene are complete homologous to this section.Here the sequence of the wild-type allele of usefulness comprises any allelotrope sequence with the target gene of curee's disease independent; Therefore, the polymorphism gene has a plurality of wild-type sequences.The position of section (a) sequence must comprise the part that will repair sudden change that contains of target gene.Unless target gene can not normal transcription target cell, otherwise the sequence of section (a) is the coding strand sequence of wildtype target gene preferably.When target gene is not transcribed in target cell, so preferably neither use the coding strand sequence also not use the noncoding strand sequence.The sequence and the bonded length of the sequence of section (a) decision section (c)-(e), section (c)-(e) must with section (a) complementation.

Section (a) and section (c) and (e) Nucleotide of the part of base pairing can be the known any 2 '-deoxyribonucleotide that maybe will develop.The section (c) and the Nucleotide (e) that are called the ribonucleotide section can be any 2 '-O-ribonucleotides, promptly contain the Nucleotide of 2 '-hydroxyl or alkyl oxide part.Here the noun of usefulness " ribonucleotide " refers to any Nucleotide with 2 '-O or 2 '-alkyl oxide.In optimum implementation, the Nucleotide that is called the section (d) that interleaves section is 2 '-deoxyribonucleotide.Section (d) also can be selected to be made up of ribonucleotide; If like this, section (c), (d) and boundary (e) are just uncertain.Section (b) and (f)-(h) can be the Nucleotide of any kind.Perhaps the connection portion between deoxyribonucleotide or ribonucleotide can be phosphodiester bond, phosphorothioate bond, phosphorodithioic acid ester bond or so any other linking group: they allow to form duplex nucleic acid, are convenient to be transfected into cell and do not disturb homologous recombination.

In optimum implementation, section (c) and sequence (e) and the complete homology of target gene.In an optimum implementation, the complete homology of wild-type allele of the sequence of section (d) and target gene; And be and one or more target mutations target gene homology in addition.

Section (b) and length (g) are approximately 4 Nucleotide, and form the hair clip turnover of strand, and described turnover makes and section (a) and (c)-(e) and section (f) and (h) formation Watson-Crick base pair promptly forms duplex nucleic acid.

Section (c) and (e) also claim the first and second ribonucleotide sections in an optimum implementation, is made up of 2 '-O-methyl ribonucleotides.In an optimum implementation, section (c) and length (e) 6-13 the Nucleotide of respectively doing for oneself.

Section (d) is also referred to as and interleaves section, and in one embodiment, its length is 4-20 Nucleotide.Section (d) must comprise the fragment homology of the point mutation that will repair with target gene, just says that in this case the described section that interleaves crosses over this sudden change.Section (d) preferably comprises 3 ' and 5 ' end of point mutation Nucleotide, just says that in this case the described section that interleaves comprises this point mutation.If target gene includes by being less than two or more point mutation that 15 Nucleotide separate, each point mutation can be repaired with same CRV so.

Section (f) and (h) form duplex, make at section (a) and the CRV 3 ' of otch is arranged (h) and 5 ' end arranged side by side.In one embodiment, 3 ' end and 5 ' end can be dephosphorylations.In selectable embodiment, 3 ' and 5 ' end can be covalently bound by phosphodiester bond or the key suitable with it, makes that CRV is the oligonucleotide of closed hoop.Can from the CRV of closed hoop, can randomly lack section (f) and (h).E. the embodiment of the present invention that relate to non-hematopoietic cell

The present invention can be used for repairing sudden change or sudden change is incorporated into and can takes out, cultivate any kind cell that also is implanted to this curee again in curee's body.Liver cell, especially liver replenish (doing) cell taking-up, cultivate and replant into technical description in Naughton, G.B. and Sibanda are B.1994 among the patent specification WO 94/08598 on April 28, in.Can comprise by the genetic diseases of repairing hepatocellular sudden change treatment: the familial hypercholesterolemia that causes by the ldl receptor sudden change; The pulmonary emphysema that the alpha1-antitrypsin transgenation causes; Reach the hemophilia and the Chris Ma Sishi disease that cause by coagulation factor VIII and IX sudden change.F. specific embodiments of the present invention

In one embodiment of the invention, make up CRV, make the sequence of described CRV comprise the sequence of at least 15 nucleotide fragments of the normal allele sequence of human beta-globin gene.The sequence of the common allele of human beta-globin is referring to Lawn etc. 1980, Fig. 2 of Cell 21:647, and this sequence is attached to herein by reference.The CRV that comprises the beta-globin fragment sequence can be used to repair the sudden change that causes sickle-cell disease, HbC and β-thalassemia.

The embodiment that is suitable for repairing the sudden change of HbC and sickle-cell disease comprises the sequence of at least 15 nucleotide fragments in the following sequence: 5 '-CAC CTG ACT CCT GAG GAGAAG TCT GCC-3 ' (SEQ ID NO:1).The embodiment that is suitable for repairing the HbE sudden change comprises the sequence of at least 15 nucleotide fragments in the following sequence: 5 '-GAA GTT GGT GGTGAG GCC CTG GGC AGG-3 ' (SEQ ID NO:2).The following stroke horizontal line of the Nucleotide that in HbC, HbS and HbE, suddenlys change.

In second embodiment of the present invention, make up CRV and make described CRV sequence comprise the sequence of at least 15 nucleotide fragments of people's glucose cerebrosidase gene normal allele sequence.The modal allelotrope sequence of glucose cerebrosidase is seen Tsuji etc., 1986, and Fig. 2 of J.Biol.Chem 261:50-53, this sequence is attached to herein by reference.

Following table I is included in the position of the more dominant sudden change of finding in familial splenic anemia and the β-thalassemia, described sudden change and is used for the table look-up that CRV repairs the wild-type allele sheet sequence of described sudden change.The sequence of table I is the sequence with 5 ' of routine → coding strand that 3 ' direction provides.About position of described sudden change shows with underscore Nucleotide.

VI. EXAMPLE Example 1. is used CRV and is repaired the additive type alkaline phosphatase

Obtained to comprise the expression plasmid of human liver/bone/kidney alkaline phosphatase cDNA, described cDNA is controlled by the SV40 early promoter, this plasmid called after pHAP.The identical plasmid and the called after p711 of this cDNA mutant form have been obtained.Fig. 2 A illustrates the design that is used for the CRV that pHAP and p711 sequence change mutually.Design CRV Ch1 is to repair the 711st missense mutation.Has a G residue corresponding to this mutational site in the wild-type sequence.The design of Ch2 is identical with Ch1, just replaces G corresponding to position 711 by A.The sequence of Ch3 and Ch1 is identical, and just the sequence of ribonucleotide section is the sequence of alkaline phosphatase genes encoding chain, rather than the sequence of noncoding strand.Oligonucleotide Dh1 comprises the sequence identical with Ch1, just only contains 2 '-deoxynucleotide.

The diagram of p711 shows the early promoter (P of single point sudden change A, the SV40 of the 711st of alkaline phosphatase cDNA coding region in Fig. 2 B _E), SV40 replication orgin (ori), polyadenylic acid add the angle of striking and be used for the little t intron sequences (SV40 polyA) of montage.Frame of broken lines among Fig. 2 B shows pBR322 coding replication orgin and β-Nei Xiananmei (Amp ^R) sequence of gene.With p711 transfection CH cell, after 6 hours, with CRV, the Ch1 Chinese hamster ovary celI of p711 of having introduced transfection in advance.Two kinds of transfections are all carried out with lipofection.The degree that changes into the wild-type phenotype is monitored on biological chemistry and two levels of dna sequence dna by spectrophotometry, histochemical stain and Hirt DNA analysis.Material and method

Synthetic and the purifying of oligonucleotide: on ABI 394 DNA/RNA synthesizers, synthesize described chimeric oligonucleotide in 0.2 μ mole scope with the wide hole CPG of 1000 .(exocyclic amino CA) is with benzoyl protection VITAMIN B4 and cytosine(Cyt), with isobutyryl protected bird purine for Applied Biosystems, Foster City for the DNA phosphoramide.(GlenResearch, Sterllng VA) with benzene oxygen ethanoyl protection VITAMIN B4, with dimethyl carbonamidine protected bird purine, protect cytosine(Cyt) with isobutyryl to 2 '-O-methyl RNA phosphoramidite.After synthetic the finishing, by at ethanol: removed the base protecting group in 20 hours in 55 ℃ of heating in the dense ammonium hydroxide (1: 3).The crude product oligonucleotide mixes with 7M urea and 10% glycerine, is heated to 70 ℃, and application of sample is on the polyacrylamide gel that contains 7M urea.After the gel electrophoresis, the DNA band shows with uv irradiating (shadowing), downcuts band, crushing and (10mM Tris-Hcl and 1mM EDTA, wash-out spends the night while shaking in pH7.5) at the TE damping fluid from glue.Contain the elutriant of gel piece with the centrifugal filter (spinfilter) of 0.45 μ m (Millipore, Bedford, MA) centrifugal, and use ethanol sedimentation.(IN) further desalination finds that the oligonucleotide of 95% above purifying is a total length to sample for Boehringer Mannheim, Indianapolis with the centrifugal post of G-25 (spin column).

Transient transfection and histochemical stain: (B.R.L., Bethesda keep Chinese hamster ovary celI among DMEM MD) containing 10%FBS.By adding 10 μ g lipofection agent among the 1ml OPTIMEM that adds in every hole, carry out transient transfection.After the oligonucleotide transfection 24 hours, measure alkaline phosphatase activities.For histochemical stain, cell is used staining fluid incubation 20 minutes with 0.15M NaCl washing three times, fixes with 50% ethanol then.Staining fluid consist of the 2mg Fast Violet that is dissolved in the 50ml water, 2ml Naphtol AS-MX alkaline phosphatase salts solution (Sigma Chemical Company, St.Louis, MO).

The active spectrophotometry of alkaline phosphatase: by (B.R.L, Betbesda MD) add 1 * 10 with the OPTIMEN that contains 100 α l of 1 μ g lipofection agent with 1 μ g plasmid p711 ⁴In the individual Chinese hamster ovary celI, on 96 orifice plates, carry out transient transfection with three parts.After 6 hours, Ch1 or other CRV of difference amount mixed with the 100 μ l OPTIMEM that contain 1 μ g lipofection agent, be added to again in each hole.After 18 hours, the sucking-off substratum, every hole adds the 200 μ l DMEM that contain 10%FBS.After the chimeric oligonucleotide transfection 24 hours, measure the alkaline phosphatase activity.(B.R.L, Betbesda MD) carry out spectrophotometry with Elisa Amplication System.Cell washs three times with 0.15M NaCl, cracking in 100 μ l NP40 damping fluids, and described NP40 damping fluid contains 10mM NaCl, 0.5%NP40,3mM MgCl ₂Tris-HCl with 10mMpH7.5.Part cell pyrolysis liquid (20 μ l) and 50 μ l Elisa substrates and 50 μ lElisa amplification agent (B.R.L, Betbesda, MD) incubations.After 5 minutes, add 50 μ l 0.3M H with the amplification agent incubation ₂SO ₄Stopped reaction.The degree of reaction is carried out in the linearity range of detection method.Under the 490nm wavelength, (B.R.L, Betbesda MD) read absorption value to read the plate instrument with Elisa.

The segmental direct dna sequencing of Hirt DNA separation, colony hybridization and PCR: after 24 hours, harvested cell is to pass through improved alkaline lysis carrier of separating DNA with the chimeric oligonucleotide transfection.In containing 50mM Tris-HCl, the 10 μ l solution of pH 8.0,10mM EDTA and contain 5mM Tris-HCl in the 110 μ l solution of the RNA enzyme A of pH 8.0,10mM EDTA and 10 μ g/ml, make cellular segregation by trysinization, washing, resuspending.Add isopyknic cell pyrolysis liquid (0.2N NaOH and 1%SDS), and then add 100 μ l neutralization solutions (3MKAc, pH 5.5) again.Behind the room temperature incubation 10 minutes, with 10, centrifugal 10 minutes of 000rpm.Supernatant liquor is used ethanol sedimentation with isopyknic phenol-chloroform extracting.With Hirt DNA be transformed into e.colidh5 (B.R.L, Betbesda, MD) in.Just each probe specificity hybridization aspect screening Hirt DNA bacterium colony of design suddenlys change with discrimination points.Allow bacterium colony on the penbritin flat board, grow, xerox on the nitrocellulose filter paper, treatedly be used for colony hybridization with double.End-labelled oligonucleotide probe 711-A of described trace and P32-(5 '-CCGCCTACACCCACTCG-3 ' (SEQ ID NO:3)) or 711-G (5 '-CCGCCTACGCCCACTCG-3 ' (SEQ ID NO:4)) are hybridized in 37 ℃ of solution that containing 5 * Denhardts, 1%SDS, 2 * SSC and 100 μ g/ml sex change salmon sperm DNAs.Trace is in 52 ℃ of washings in TMAC solution (3.0M tetramethylammonium chloride/50mM Tris-HCl, pH 8.0,2mM EDTA and 0.1%SDS).(Chatworth, CA), 20 bacterium colonies of hybridizing from demonstration and 711-G or 711-A prepare plasmid DNA to use Qiagen to prepare test kit in a small amount.(Foster City's hundreds of base of the 711st both sides of each plasmid CA) checks order from both direction for ABI 373A, Applied Biosystem by automatic sequencing.Employing corresponding to two primers of the 630-650 position and the 803-822 position of the 711st both sides of described alkaline phosphatase cDNA (5 '-CAATGTCCCTGATGTTATGCA-3 ' (SEQ ID NO:5) and 5 '-CGCTGGGCCAAGGACGCT-3 ' (SEQ ID NO:6), use Vent _RPolysaccharase (New England Biolabs, Beverly, MA) pcr amplified fragment of 190 bp of generation.With described amplified fragments gel-purified, carry out then automated DNA order-checking (ABI 373A, Applied Biosystem, Foster City, CA).

The detection of oligonucleotide stability: with 10ng ³²The end-labelled oligonucleotide of P-mixes with the unmarked few nucleic acid of 500ng, and as above carries out transfection.For reducing the nonspecific combination of oligonucleotide, cell thoroughly washs with the solution that contains 1M NaCl/HAc pH2.5 with PBS.Containing 10mM Tris-HCl pH7.5,0.5mM MgCl ₂With lysing cell in the solution of 0.5%Trito X-100, prepare thick lysate, use phenol-chloroform extracting then.Lysate coagulates with 15% polyacrylamide that contains 7M urea to be analyzed, and then carries out radioautograph.The oligonucleotide of incubation is handled and is analyzed with the same manner in containing the DMEM of 10%FBS.

In our experimental design, various chimeric oligonucleotides are introduced the Chinese hamster ovary celI of using the p711 transfection in advance.The degree that changes into the wild-type phenotype is monitored with histochemical stain; Red pigment is deposited on represents to have organized enzyme on the cell.When the cell that contains mutator gene was used 11nM Ch1 transfection, the frequency averaging that red cell occurs almost accounted for and is subjected to 1/3 of transfection CHO cell.On the contrary, Ch2 and Dh1 do not cause that enzymic activity increases.When cell was used the Ch3 transfection, it was low to observe the frequency that changes into wild-type.It is 30% that the transfection efficiency that expression by wild plasmid pHAP records is estimated.

Enzymic activity is also used above-mentioned spectrophotometry.When the p711 plasmid existed, the dose-dependently of alkaline phosphatase activities increased when observing height to 17nM Ch1.With the cell that 17nM Ch1 handles, its enzymic activity is near using 60% of wild plasmid pHAP transfectional cell enzymic activity.This increase is sequence-specific, because the Ch1 of equivalent does not influence the enzymic activity with the pHAP transfectional cell.In addition, the Ch2 that only differs from a base pair with sequence C h1 does not cause that enzymic activity increases.The oligonucleotide Dh1 that contains the sequence identical with Ch1 but do not contain the ribonucleotide section shows that enzymic activity does not increase.Therefore, alkaline phosphatase enzymic activity spectrophotometry is consistent with the result of histochemical staining method.

Point mutation with chimeric oligonucleotide testing target dna sequence dna: in order to confirm the variation of dna sequence dna level, in chimeric oligonucleoside transfection after 24 hours, with improving alkaline lysis (Wang, G. etc., 1995, Mol Cell Biol.15,1759), from preparing the Hirt extract with p711 and various oligonucleotide cells transfected.Hirt DNA transforms DH5 α cell effectively, from 10 ⁶The individual Chinese hamster ovary celI of transfection that is subjected to produces 10 ⁴Individual Amp ^RThe mattress bacterium.In screening DH5 α transformant aspect the probe specificity hybridization that designs discrimination points sudden change (A) and wild-type (G) sequence, described point mutation sequence and wild-type sequence are respectively corresponding to the 703-719 position of cDNA with normal cDNA that suddenly change, Weiss, MJ., 2988, Proc.Natl Acad.Sci 85:7666.From at least twice more than 500 mattress bacterium of a plurality of flat boards of producing of transfection experiment independently, by measuring correction rate (table I) with the mean number of the bacterium colony of 711-G probe or 711-A probe hybridization.Two crowdes of Ch1 by independent synthetic preparation observe close transformation efficiency.The nearly 70% mattress bacterium and the 711A probe hybridization that produce from Hirt DNA with the preparation of p711 and Ch1 transfectional cell, and 30% bacterium colony shows and 711-G probe hybridization (table I).Therefore, but under 11nM Ch1 the correction rate of repeated observation to 30%.Hybridization is special, does not observe cross hybridization between two kind of groups.Application is carried out dna sequencing from both direction with the plasmid that 20 bacterium colonies these bacterium colonies prepare corresponding to two primer 5 '-CAATGTCCCTGATGTTATGCA-3 ' (SEQ ID NO:7) and 5 '-CGCTGG GCCAAGGACGCT-3 ' (SEQ ID NO:8) of the 630-650 position and the 803-822 position of the 711st both sides of described alkaline phosphatase cDNA.All confirm the sequence conversion in each case, and in hundreds of bases around the target nucleotide, do not observed other sequence change.Come since Ch2 or Dh1 handle cell preparation the Hirt extract all bacterium colonies only with 711-A probe hybridization (table I).From some bacterium colony and the wild-type probe hybridization of the Hirt extract of Ch3, but its degree is than Ch1 little a lot (table II).These results confirm that the different alkaline phosphatase activitieses of demonstration are owing to the sudden change of check point on the dna sequence dna level (A → G) produce.

The table II. from the dual transfection preparation of the various oligonucleotide of p711 plasmid and 11nM

The crossing pattern of the transformant that obtains of Hirt extract

Oligonucleotide	The transfection number	The total number of bacterial colony of every flat board	Colony number with 711-G hybridization	Colony number with 711-A hybridization	Transform %
						????Ch1	????1	????84 ????189 ????219	????32 ????70 ????74	????54 ????117 ????143	????38 ????37 ????34
????2	????139 ????162 ????159	????42 ????49 ????51	????98 ????110 ????108	????30 ????30 ????32
					????Ch2		????1	????108 ????90	????0 ????0	????108 ????90	????0 ????0
????2	????218 ????148	????0 ????0	????218 ????148	????0 ????0
						????Ch3	????1	????190 ????151	????3 ????4	????185 ????145	????2 ????3
????2	????189 ????143	????0 ????0	????185 ????143	????0 ????0
					????Dh1		????1	????217 ????180	????0 ????0	????217 ????180	????0 ????0
????2	????157 ????188	????0 ????0	????157 ????188	????0 ????0

Being used to breed the RecA defective escherichia coli bacterial strain of plasmid DNA can enough additive type DNA (21) repair and carries out autosyndetic pairing.In order to get rid of the sequence conversion is the possibility that is mediated by intestinal bacteria, carries out the direct dna sequencing of Hirt DNA pcr amplified fragment.By the effect of VentR polysaccharase, produce the pcr amplified fragment of 190bp with two primers of the 711st both sides.The result shows, when the transfectional cell from the composition of p711 and Ch1 prepared Hirt DNA sample, the 711st was A (70%) and the mixture of G (30%).On the contrary, when when oligonucleotide Dh1 prepares Hirt DNA, do not observe mixed sequence at 711.These results have confirmed that clearly the sequence correction of carrying out with chimeric oligonucleotide takes place in mammalian cell.

The stability of chimeric oligonucleotide: measure in the born of the same parents and contain the stability of chimeric oligonucleotide described in the growth medium of 10%FBS.The radiolabeled oligonucleotide Ch1 of 10ng is added in the identical transfection experiment that carries out histochemical stain and Hirt DNA analysis (referring to reference material and method).Described chimeric oligonucleotide is extremely stable.When chimeric oligonucleotide in the growth medium that contains 10%FBS during incubation, incubation was not observed detectable degraded after 24 hours.In addition, at same incubation time durations, do not show any degraded from the oligonucleotide of cellular segregation yet.When incubation after 24 hours during, only detect the monomer of chimeric oligonucleotide from cellular segregation.Therefore, here under the experiment condition of usefulness, the head and the tail of not observing chimeric oligonucleotide connect (Litigation).Embodiment 2. usefulness CRV repair the beta-globin gene of EB-transformant

Designed the CRV that is used for repairing the sudden change of finding at sickle-cell disease beta-globin gene, i.e. the SC1 of Fig. 3.This molecule is made up of the DNA residue that contains inserted block between two, and 10 2 '-O-methyl RNA residues of the short sequence flank that described inserted block is 5 DNA are formed.When this molecular folding was the duplex conformation, a chain only contained the DNA residue, and another key contains the RNA/DNA piece.Like this, internal sequence and β ^SCross over β in the-globin sequence ^SExcept the sequence complementation on 25 residue sequence in mutational site, runic and single base (T) of marking with an asterisk.Flank is that the center of the RNA of 5 DNA residues approximately is arranged in β S encoding sequence mutation T residue.Except the base (A) that indicates runic and asterisk, designed contrast chimeric oligonucleotide (SC2) with the same manner.β ^A, β ^SBe also shown among Fig. 3 A β with the genome sequence of closely-related δ-globin gene ^SThe specific site of sudden change prints with runic.

Be prepared as follows the lymphoblastoid cell.From sickle-cell disease patient and one both do not had this disease medical history also asymptomatic investigator's disposable clinical material obtain the blood that heparin is handled.By in Ficoll, carrying out density gradient centrifugation, obtain monocyte from blood (≈ 8ml), and be used in the ebv infection that the marmoset MC is breeding among the B95-8 (Coriell Intitute for Medical Research#GM07404D).In the T25 flask, added the leucoagglutinin PHA-L that adds 0.1mg in the RPMI substratum of 20% foetal calf serum at 10ml and infected.Since the 5th day, send nutriment to culture 2 times weekly, set up culture in case in the time of the 21st day, there is the cell of 60-70% to keep survival just to think.β ^AAnd β ^SLymphoblastoid keeps in containing the RPMI substratum of 10% foetal calf serum.

Following described CRV is incorporated into β ^SIn the above-mentioned lymphoblastoid that allelotrope isozygotys.Test the day before yesterday, in 24 hole tissue culturing plates, every hole 1ml culture medium inoculated cell 1 * 10 ⁵Individual cell/ml.By with chimeric oligonucleotide with contain 3mg DOTAP (N-[1-(2,3-two oily acyloxy) propyl group]-N, N, N-TMA (TriMethylAmine) mesylate, 20ml 20mM HEPES Boehringer-Mannheim), pH 7.3 mixes, in room temperature incubation 15 minutes, be added to then and carry out transfection in the cultured cells.After 6 hours, centrifugal cell harvesting, washing, and preparation is used for method (the PCR Protocols by E.S.Kawasaki, editor M.A.Innis, D.H.Gelford, J.J.Sninsky and T.J.White, 146-152 page or leaf, Acadamic Press, 1990) carry out pcr amplification.

Utilization know by β ^SThe correction of the restriction fragment length polymorphism valuation form base mutation that causes of sudden change, referring to R.F.Greeves etc., 1981, Proc.Natl.Acad.Sci.78:5081; J.C.Chang and Y.W.Kan, 1982, N.Eng.J.Med.307:30; S.H.Orkin etc., ibid, the 32nd page; J.T.Wilson etc., 1982, Proc.Natl.Acad.Sci.79:3628).β ^SThe transversion of T → A causes the forfeiture of Bsu36I restriction site (CCTGAGG) in the allelotrope.Therefore, the genomic dna Southern hybridization analysis cut of Bsu36I enzyme can detect β ^SAllelotrope.The 1.2kbp Bsu36IDNA fragment of the beta-globin gene of normal presence is at β ^SDo not exist in the allelotrope, and replace by the diagnostic fragment of 1.4kpb.As β from isozygotying ^SWhen the genomic dna that reclaims in the lymphoblastoid is analyzed in this way, observe the 1.4kbp of expection.Yet, in DNA, observe two fragments with SC1 CRV transfectional cell.Except the 1.4kbp fragment, also there is the 1.2kbp fragment, shows β ^SAllelic part is proofreaied and correct and is carried out in the dose-dependently mode.

In order to measure correction rate fast, delicately, we adopt the rflp analysis of PCR-based.In order to analyze the beta-globin gene order, use primer BG02 (5 '-TCCTAAGCCAGTGCCAGAAGA-3 ' (SEQ ID NO:9)) and BG05 (5 '-CTATTGGTCTCCTTAAACCTG-3 ' (SEQ ID NO:10)) and amplification Taq polysaccharase (BoehringerMannheim), from the PCR fragment of granular cell lysate amplification preparation 345bp.In order to analyze δ-globin gene, in amplified reaction,, produce the fragment of a 335bp with identical cell extract and primer DG06 (5 '-CTCACAAACTAATGAAACCCTGC-3 ' (SEQ ID NO:11)) and DG07 (5 '-GAAAACAGCCCAAGGGACAG-3 ' (SEQ ID NO:12)).Gel dyes with SYBRTM green (FMC Bioproducts), and fluorescence intensity is quantitatively next with the fluorescence imaging device of Molecular Dynamics.On both direction, carry out dna sequencing with ABI 373A sequenator.

Primer above the design is crossed over β to produce one behind the pcr amplification genomic dna ^S345 bp fragments in mutational site.Normocellular fragment comprises the Bsu36I recognition sequence, and produces the fragment of 228bp and 117bp, and from β ^SThe DNA of DNA comprises sequence C CTGTGG, and is difficult to enzyme and cuts.Analysis is pointed out from the β of SC1 processing ^SThe 345bpDNA fragment of cell amplification is partially digested with Bsu36I, has shown that the correction of sudden change just takes place on some karyomit(e)s, is not to take place on all karyomit(e)s.Also use fluorescence dye SYBR after the electrophoretic separation ^TMAfter the green dyeing, by the relatively relative intensity acquisition quantitative measurment of three dna fragmentations.Make the colour band imaging with the laser fluorescence imager, and calculating their level relatively.By scanning the painted sepharose of cyber green, quantitative assay transformation efficiency with the fluorescence imaging device.Dosage is 2.5-25.0pM SC1/10 ⁵Individual β ^SThe experiment of lymphoblastoid shows, β ^S→ β ^ATransformation efficiency be approximately 40-50% (Fig. 4 A).

The sickleshaped sudden change is measured with the method that proposes above by the pull-in frequency of CRV SC2.Analysis revealed, under the highest level 25nM of input chimeric molecule, level of corrections surpasses 50%, even but under 2.5nM, observe 30% beta-protein gene and also obtain proofreading and correct (Fig. 4 B).

The segmental direct order-checking of the 345bp of pcr amplification confirms the change of T → A in coding strand.From β ^SThe DNA sample in, cell is with being higher than 12nM/10 ⁵The chimeric molecule SC1 transfection of the higher concentration of individual cell.Sequential analysis has shown at β ^SA and the almost equal mixture of T residue on the mutational site.When handling with SC1, untreated β ^SThe DNA of cell only contains T in this site, and β ^AThe DNA of cell only contains A.β with contrast CRV SC2 transfection ^SThe processing of cell does not cause the change of beta-globin gene order.Yet, proved that as this sequence mixture by T and A residue on the sequence site of expection the Normocellular DNA of SC2 transfection partly changes into β ^SMutant nucleotide sequence.

The specificity of the order-checking appraisal CRV effect by relevant δ-globin gene, described relevant δ-globin gene are more than 90% and the beta-globin dna homolog.β is identical on the core target area of SC15bp DNA with the δ globin gene.In Fig. 3 at two different single base underscores.In order to determine whether SC2 has changed δ-globin gene, has as above carried out sequential analysis.The result shows: with observed by SC2 at β ^AThe change of instructing in-globin the sequence is opposite, and SC2CRV does not introduce in δ-globin gene and changes.Embodiment 3.CRV repairs the experiment usage material and the method for the beta-globin gene of HSC

Stem cell separates and transfection: continuous 5 days one day twice subcutaneous giving and normal volunteer G-CSF 300 μ g.At the 4th day and the 5th day of the G-CSF treatment, they utilized the COBE wave spectrum to remove instrument (spectra pheresis machine) through the 4 hours single blood sampling composition of stem cell arts.By (density 1.077g/ml, Pharmacia) density gradient centrifugation on (2000rpm, 10 minutes, room temperature) prepares monocyte at Ficoll-Hypaqne.Remove after adhering on the plastics most of monocyte (30 minutes, 37 ℃, 5%CO ₂, contain the RPMI of 10%FBS).To take out the loose cell that sticks on the plastics, harvested cell is with PBS washing three times by rotation.In room temperature incubation 25 minutes, concentration was 100 * 10 in the PBS/1%BSA that contains biotinylation mouse-anti CD34 antibody in this colony ⁶Individual cell/ml.The cell that antibody treatment is crossed passes through the avidin post, collects and analyzes by the cell of pillar.Wash pillar with PBS subsequently, stick to the CD34 on the pillar ⁺Cell reclaims by the extruding pillar.Last purity is evaluated by FACS.

With the cell resuspending in the RPMI that contains 10% heat inactivation FCS, with 1 * 10 ⁵Individual cell/ml paves plate in 24 orifice plates, and every hole accepts 1 * 10 ⁵Individual cell.With shown in the chimeric oligonucleotide of amount mix with 3 μ g DOTAP among the 20 μ l 20mM pH7.3 HEPES.Mixture ice bath 15 minutes is added in the cell then.In 37 ℃ of 5%CO ₂Under cultivate 16 hours after, harvested cell with its precipitation, with PBS washing, and is used the lysis buffer cracking.

Pcr amplification and analysis. use PCD2 (5 '-TCCTAAGCCAGTGCCAGAAGA-3 ' (SEQ ID NO:13)) and PCO5 (5 '-CT ATTGGTCTCCTTAAACCTG-3 ' (SEQ ID NO:14)) and amplification Taq polysaccharase (Boehringer Mannheim respectively, Indianapolis, IN) in the reaction of 50 μ l, in 94 ℃ 30 seconds, in 52.5 ℃ 30 seconds, carried out 35 circulations in 30 seconds in 72 ℃, the pcr amplification genomic dna, the fragment of a 345bp of generation.For to the δ locus, 5 ' primer is 5 '-CTCAC AAACCTAATGAAACCCTGC-3 ' (SEQ ID NO:15), 3 ' primer is 5 '-GAA AACAGCCCAAGGGACAG-3 ' (SEQ ID NO:16), in 94 ℃ 30 seconds, in 59 ℃ 30 seconds, in 72 ℃ 30 seconds, carry out 35 circulations.

The PCR product with DdeI or BSU36I restriction endonuclease (New EnglandBiolabs, Beverly, MA) digestion, (1 * TBS) carries out electrophoresis to application of sample to 1.2% sepharose.This gel is in containing 1: 20,000 cyber green strain (dark place dyeing is 20 minutes among 200ml 1 * TBE ME) for FMC, Rockland, by the fluorescence imaging device (MolecularDynamics, Sunnyvale, CA) quantitative.The PCR product by the centrifugal post of Qiaquick PCR purifying (purification spin colume) (Qiagen, Chatsworth, CA) centrifugal in water, vacuum-drying to 5 μ l, and with its concentration of O.D spectral photometry of 260nm.DNA sample (30 μ g) is by automatic Applied Biosystems Model 373A dna sequencing system (AppliedBiosystems, Foster Cify, CA) directly order-checking.

Synthetic and the purifying of oligonucleotide: adopt the wide hole CPG of 1000 , on ABI 394DNA/RNA synthesizer, synthesize chimeric oligonucleotide in 0.2 μ mol scope.In this formation thing, the exocyclic amino of DNA phosphoramidite (Applied Biosystem) is with benzoyl protection VITAMIN B4 and cytosine(Cyt), with isobutyryl protected bird purine.(Glen Research Sterling.VA) with benzene oxygen ethanoyl protection VITAMIN B4, with dimethyl carbonamidine protected bird purine, protects cytosine(Cyt) with isobutyryl to 2 '-methoxyl group RNA phosphoramidite.After synthetic, by at ethanol: removed the base protecting group in 20 hours in 55 ℃ of heating in the dense ammonium hydroxide (1: 3).By polyacrylamide gel electrophoresis purifying crude product oligonucleotide, this sample mixes with 7M urea element and 10% glycerine, is heated to 70 ℃, is added to then on the polyacrylamide gel that contains 7M urea element.After the gel electrophoresis, make the DNA band as seen, downcut the DNA band, crushing and shaken overnight wash-out in TE damping fluid (10mM Tris-HCl and 1mM EDTA pH7.5) from this gel by the UV irradiation.The elutriant that contains gel pieces by 0.45 μ m centrifugal filter (Millipore, Bedfod, MA) centrifugal, and use ethanol sedimentation.Sample is in further desalination of the centrifugal post of G-25 (Boehringer Mannheim), and finds that the purification of oligonucleotides more than 95% is a total length.The result

Isolating CD34 ⁺Enrichment colony at first is used for the oligonucleotide absorption experiment.Except radio-labeling places 5 ' end of this oligonucleotide, under these conditions chimeric molecule SC2 is mixed with Liposomal formulation DOTAP.With the mark of increasing amount with unlabelled oligonucleotide and described liposome incubation 15 minutes.This mixture and cell incubation are after 6 hours then, and cell is with the thorough wash-out of PBS, to reduce non-specific binding.Eccentric cell precipitates part with 0.2M glycerine (pH4.5) washing, to remove residual non-specific binding then.With the radioactivity in the scintillation counting mensuration cell precipitation.Cell absorbs described chimeric oligonucleotide in the dose-dependently mode.Because our experimental strategy concentrates on the nM concentration, so we can not extend beyond 25nM with curve.Ratio based on radiolabeled chimeric oligonucleotide is lived, and supposes that each cell has same ability to accept to conversion, and we estimate the CD34 to about 50% ⁺Cell colony is subjected to this substrate transfection.For each experiment, background level can by radiolabeled chimeric molecule when not having DOTAP and cytomixis evaluate, and this level never can surpass 0.05%.

Contain β with the SC2 of difference amount and the DOTAP transfection of 3 μ g/ml ^AGenotypic two allelic CD34 ⁺Enrichment of cell colony.As above-mentioned transfection after 16 hours, isolation of genomic DNA, β ^A→ β ^STransforming degree by the Restriction Enzyme polymorphism and directly dna sequencing measure.From 10 ⁵The gene DNA of individual cellular segregation carries out pcr amplification with two primer PCO2 and PCO5, produces the fragment of a 345bp.Cut β with restriction enzyme DdeI enzyme ^ASpecific sequence produces three fragments that are respectively 192bp, 108bp and 45bp, and β ^SA sequence enzyme is cut once, produces the fragment of 300bp and 45bp.Observing the segmental level of 300bp that enzyme not cuts increases with the increase of SC2 concentration, shows β ^A→ β ^SGenotypic conversion is referring to Fig. 5.(observe 50% transformation efficiency under the chimeric oligonucleotide of 600ng=30nM * 1ml) at relative lower concentration.On the contrary, do not observe conversion in the cell of handling with SC1, SC1 is and β ^AThe complete complementary chimeric molecule of site paired.

For the change (A → T), carry out the segmental direct dna sequencing of 345bp that proves dna sequence dna in normal cell.Contain the β that isozygotys as above-mentioned with 23nM SC2 transfection ^AAllelic CD34 ⁺Colony.Isolation of genomic DNA carries out pcr amplification, then sample is carried out the automated DNA order-checking.Independent β ^AThe β that dna sequence dna and SC1 handle ^ADna sequence dna all comprises T.On the contrary, the β that handles with SC2 ^AThe dna sequence dna of cell shows that on desired location the dose-dependently of A → T transforms.SC2 CRV comprises (a) section identical with beta-globin genes encoding chain.The CRV of called after SC5 comprises (a) section identical with the noncoding strand fragment of beta-globin gene.We repeat above-mentioned transfection experiment with SC2 and SC5.As if the results are shown in Figure 5, although show that not have SC2 active strong, SC5 is activated, be lower than under the 20nM non-activity.

From the β that has handled with SC2 ^AThe genomic dna of cell is with two δ-globin special primer PCO ₆And PCO ₇Carry out pcr amplification.Only found wild-type δ-globin sequence, this has proved that SC2 CRV is specific to beta-globin.

The table I

Beta Thalassemia
					Codon	Base substitution	Affected ethnic group	Target sequence is rule under influenced sequence or codon	SEQ?ID?NO:
Non-functional mRNA nonsense mutant
					????17	????A→T	Chinese	GCC?CTG?TGG?GGC?AAG?GTG?AAC?GTG?GAT	????17
????39	????C→T	Mediterranean Sea/European	TAC?CCT?TGG?ACC?CAG?AGG?TTC?TTT?GAG	????18
					????15	????G→A	The Indian	GTT?ACT?GCC?CTG?TGG?GGC?AAG?GTG?AAC	????19
????121	????A→T	Pole/Swiss	CAC?TTT?GGC?AAA?GAA?TTC?ACC?CCA?CCA	????20
					????37	????G→A	Saudi Arabian	GTG?GTC?TAC?CCT?TGG?ACC?CAG?AGG?TTC	????21
????43	????G→T	Chinese	CAG?AGG?TTC?TTT?GAG?TCC?TTT?GGG?GAT	????22
					????61	????A→T	Black people	AAC?CCT?AAG?GTG?AAG?GCT?CAT?GGC?AAG	????23
????35	????C→A	Thailander	CTG?CTG?GTG?GTC?TAC?CCT?TGG?ACC?CAG	????24
Non-functional mRNA phase shift mutant
					????1	-G disappearance	People from Mediterranean Sea	ACA?GAC?ACC?ATG?GTG?CAC?CTG?ACT?CCT	????25
????5	-CT disappearance	People from Mediterranean Sea	GTG?CAC?CTG?ACT?CCT?GAG?GAG?AAG?TCY	????26
					????6	-A disappearance	People from Mediterranean Sea	CAC?CTG?ACT?CCT?GAG?GAG?AAG?TCY?GCN	????27
????8	-AA disappearance	The Turk	ACT?CCT?GAG?GAG?AAG?TCY?GCN?GTT?ACT	????28
					????8/9	+ G inserts	The Indian	ACT?CCT?GAG?GAG?AAG?TCY?GCN?GTT?ACT	????29
????11	-T disappearance	The Mexican	GAG?AAG?TCT?GCC?GTT?ACT?GCC?CTG?TGG	????30
					????14/15	+ G inserts	Chinese	GCC?GTT?ACT?GCC?CTG?TGG?GGC?AAG?GTG?AAC	????31
????16	-C disappearance	The Indian	ACT?GCC?CTG?TGG?GGC?AAG?GTG?AAC?GTG	????32
					????27-28	+ C inserts	Chinese	GTT?GGT?GGT?GAG?GCC?CTG?GGC?AGG?CTG?CTG	????33
????35	-C disappearance	The Indonesian	CTG?CTG?GTG?GTC?TAC?CCT?TGG?ACC?CAG	????34
					????36-37	-T disappearance	The Iranian	CTG?GTG?GTC?TAC?CCT?TGG?ACC?CAG?AGG?TTC	????35

Beta Thalassemia
					Codon	Base substitution	Affected ethnic group	Target sequence is rule under influenced sequence or codon	SEQ?ID?NO:
????37	-G disappearance	The Kurds	GTG?GTC?TAC?CCT?TGG?ACC?CAG?AGG?TTC?TTT	????36
					????41/42	-CTTT disappearance	Indian/Chinese	TGG?ACC?CAG?AGG?TTC?TTT?GAG?TCC?TTT?GGG	????37
????44	-C disappearance	The Kurds	AGG?TTC?TTT?GAG?TCC?TTT?GGG?GAT?CTG	????38
					????47	+ A inserts	The Suriname Black people	GAG?TCC?TTT?GGG?GAT?CTG?TCC?ACT?CCT	????39
????64	-G disappearance	The Swiss	GTG?AAG?GCT?CAT?GGC?AAG?AAA?GAG?CTC	????40
					????71	+ T inserts	Chinese	GTG?CTC?GGT?GCC?TTT?AGT?GAT?GGC?CTG	????41
????71/72	+ A inserts	Chinese	GTG?CTC?GGT?GCC?TTT?AGT?GAT?GGC?CTG?GCT	????42
					????76	-C disappearance	The Italian	AGT?GAT?GGC?CTG?GCT?CAC?CTG?GAC?AAC	????43
????82/83	-G disappearance	The Azerbaijanians	CTG?GAC?AAC?CTC?AAG?GGC?ACC?TTT?GCC?ACA	????44
					????94	+ TG inserts	The Italian	GAG?CTG?CAC?TGT?GAC?AAG?CTG?CAC?GTG	????45
????106/107	+ G inserts	The America Black people	AAC?TTC?AGG?CTC?CTG?GGC?AAC?GTG?CTG?GTC	????46
					????109	-G disappearance	The Lithuanians	CTC?CTG?GGC?AAC?GTG?CTG?GTC?TGT?GTG	????47
????126	-T disappearance	The Italian	TTC?ACC?CCA?CCA?GTG?CAG?GCN?GCC?TAT	????48
The initiator codon mutant
					????ATG-AGG		Chinese	CAA?ACA?GAC?ACC?ATG?GTG?CAC?CTG?ACT	????49
????ATH-ACG		Yugoslav	CAA?ACA?GAC?ACC?ATG?GTG?CAC?CTG?ACT	????50
RNA processing mutant-montage circle changes
					The 1st of IVS1	????G→A	People from Mediterranean Sea	GAG?GCC?CTG?GGC?AGG?TTG?GTA?TCA?AGG	????51
The 1st of IVS1	????G→T	Indian/Chinese	GAG?GCC?CTG?GGC?AGG?TTG?GTA?TCA?AGG	????52
					The 1st of IVS2	????G→A	People from Mediterranean Sea/Tunisian/America Black people	GAG?AAC?TTC?AGG?GTG?AGT?CTA?TGG?GAC	????53
The 2nd of IVS1	????T→G	The Tunisian	GCC?CTG?GGC?AGG?TTG?GTA?TCA?AGG?TTA	????54
					The 2nd of IVS1	????T→C	Black people	GCC?CTG?GGC?AGG?TTG?GTA?TCA?AGG?TTA	????55

1 beta Thalassemia
					Codon	Base substitution	Affected ethnic group	Target sequence is rule under influenced sequence or codon	SEQ?ID?NO:
IVS13 ' end	????G→C	The Italian	????TTT?CCC?ACC?CTT?AGG?CTG?CTG?GTG?GTC	????56
					IVS23 ' end	????A→G	The America Black people	????ATC?TTC?CTC?CCA?CAG?CTC?CTG?GGC?AAC	????57
IVS23 ' end	????A→C	The America Black people	????ATC?TTC?CTC?CCA?CAG?CTC?CTG?GGC?AAC	????58
					IVS13 ' end	????G→A	The Egyptian	????TTT?CCC?ACC?CTT?AGG?CTG?CTG?GTG?GTC	????59
					RNA processing mutant-total the variation
The 5th of IVS1	????G→C	Indian/Chinese/Melanesian	???CTG?GGC?AGG?TTG?GTA?TCA?AGG?TTA?CAA	????60
					The 5th of IVS1	????G→T	People from Mediterranean Sea/Black people	???CTG?GGC?AGG?TTG?GTA?TCA?AGG?TTA?CAA	????61
The 5th of IVS1	????G→A	The Algerian	???CTG?GGC?AGG?TTG?GTA?TCA?AGG?TTA?CAA	????62
					The-1 of IVS1 (codon 30)	????G→C	Tunisian/Black people	???GAG?GCC?CTG?GGC?AGG?TTG?GTA?TCA?AGG	????63
The-1 of 1VS1 (codon 30)	????G→A	The inferior people of Bulgaria	???GAG?GCC?CTG?GGC?AGG?TTT?GTA?TCA?AGG	????64
					The-3 of IVS1 (codon 29)	????C→T	The Lebanese	???GGT?GAG?GCC?CTG?GGC?AGG?TTG?GTA?TCA	????65
IVS23 ' end	????CAG→AAG	Iranian/Egyptian/Black people	ATC?TTC?CTC?CCA?CAG?CTC?CTG?GGC?AAC?GTG	????66
					IVS13 ' end	????TAG→GAG	Saudi Arabian	TAT?TTT?CCC?ACC?CTT?AGG?CTG?CTG?GTG?GTC	????67
					Inner intron changes
The 110th of IVS1	????G→A	People from Mediterranean Sea	TCT?CTC?TGC?CTA?TTT?TGG?TCT?ATT?TTT?CCC	????68
					The 116th of IVS1	????T→G	People from Mediterranean Sea	???TGC?CTA?TTG?GTC?TAT?TTT?CCC?ACC?CTT	????69
The 705th of IVS2	????T→G	People from Mediterranean Sea	???AAA?TTG?TAA?CTG?ATG?TAA?GAG?GTT?TCA	????70
					The 745th of IVS2	????C→G	People from Mediterranean Sea 73	AGC?AGC?TAC?AAT?CCA?GCT?ACC?ATT?CTG?CTT	????71
The 654th of IVS2	????C→T	Chinese 303	???TTT?CTG?GGT?TAA?GGC?AAT?AGC?AAT?ATT	????72

Beta Thalassemia
					Codon	Base substitution	Affected ethnic group	Target sequence is rule under influenced sequence or codon	SEQ?ID?NO:
The coding region displacement of influence processing
					????26	????G→A	The Southeast Asian	GAA?GTT?GGT?GGT?GAG?GCC?CTG?GGC?AGG	????73
????24	????T→A	The America Black people	GTG?GAT?GAA?GTT?GGT?GGT?GAG?GCC?CTG	????74
					????27	????G→T	People from Mediterranean Sea	GTT?GGT?GGT?GAG?GCC?CTG?GGC?AGG?CTG	????75
????19	????A→G	The Malaysian	CTG?TGG?GGC?AAG?GTG?AAC?GTG?GAT?GAA?GTT	????76

This information is selected from The Metabolie and Molecular Bases of Inherited Disease, Chartes R.Seriver edits (John B.Stanbury, James M.Wyngaarden, Donald S.Frederickson, the advisory editor) the 7th edition, McGraw-Hill, Health Profesions Division, New York, 1995.

Familial splenic anemia
							The cDNA numbering	The amino acid numbering	The genome numbering	Nucleotide subsitution	Amino-acid substitution	Target sequence is at the Nucleotide underscore of sudden change	SEQ?ID?NO:
					Common
							????1226	????370	????5841	????A→G	????Asn→Ser	????AGC?ATC?ATC?ACG?AAC?CTC?CTG?TAC?CAT	????77
????1448	????444	????6433	????T→C	????Leu→Pro	????CAG?AAG?AAC?GAC?CTG?GAC?GCA?GTG?GCA	????78
							????84	????1035		G → GG inserts		????CCT?AAA?AGC?TTC?GGC?TAC?AGC?TCG?GTG	????79
												Non-common
	????IVS2	????1067	????G→A		????TGT?CGT?GGG?CAT?CAG?GTG?AGT?GAG?TCA	????80
							????754	????213	????3548	????T→A	????Phe→Ile	????TGG?GCC?AGA?TAC?TTT?GTG?AAG?TTC?CTG	????81
????1192	????359	????5408	????C→T	Arg → termination	????GGC?TCC?TGG?GAT?CGA?GGG?ATG?CAG?TAC	????82
							????1193	????359	????5409	????G→A	????Arg→Gln	????GGC?TCC?TGG?GAT?CGA?GGG?ATG?CAG?TAC	????83
????1297	????394	????5912	????G→T	????Val→Len	????GGA?CCC?AAT?TGG?GTG?CGT?AAC?TTT?GTC	????84
							????1342	????409	????5957	????G→C	????Asp→His	????GAC?ATC?ACC?AAG?GAC?ACG?TTT?TAC?AAA	????85
????1504	????463	????6489	????C→T	????Arg→Cys	????GTC?CTA?CTA?AAC?CGC?TCC?TCT?AAG?GAT	????86
							????1604	????496	????6683	????G→A	????Arg→His	????TAC?CTG?TGG?CGT?CGC?CAG?TGA?TGG?AGC	????87

Sequence table (1) physical data

(ⅰ) applicant:

(A) addressee: Thomas Jeffrson University

(ⅱ) denomination of invention: the method and the compound of the disease that the treatment sudden change causes

(ⅲ) sequence number: 100

(ⅳ) mailing address:

(A) addressee: Pennie ﹠amp; Edmonds

(B) street: 1155 Avenue of the Americas

(C) city: New York

(D) state: New York

(E) country: the U.S.

(F) postcode: 10036-2711

(ⅴ) computer-reader form

(A) medium type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, version 1.30

Ask data in (ⅵ) current

(A) application number: PCT/US97/

(B) submission date: on May 1st, 1997

(C) classification:

(ⅶ) request for data formerly

(A) application number:

(B) submission date:

(ⅷ) attorney/proxy's data

(A) name: Friebel, Thomas E

(B) number of registration: 29,258

(C) reference/file number: 7991-011-228

(ⅸ) telecommunication data

(A) phone: (212) 790-9090

(B) fax: (212) 869-9741/8864

(C) telegram: the information of 66141 PENNIE (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:1:CACCTGACTC CTGAGGAGAA GTCTGCC 27 (2) SEQ ID NO:2:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:GAAGTTGGTG GTGAGGCCCT GGGCAGG 27 (2) SEQ ID NO:3:

(ⅰ) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3:CCGCCTACAC CCACTCG 17 (2) SEQ ID NO:4:

(ⅰ) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4:CCGCCTACGC CCACTCG 17 (2) SEQ ID NO:5:

(ⅰ) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5:CAATGTCCCT GATGTTATGC A 21 (2) SEQ ID NO:6:

(ⅰ) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:CGCTGGGCCA AGGACGCT 18 (2) SEQ ID NO:7:

(ⅰ) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:7:CAATGTCCCT GATGTTATGC A 21 (2) SEQ ID NO:8:

(ⅰ) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:8:CGCTGGGCCA AGGACGCT 18 (2) SEQ ID NO:9:

(ⅰ) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:9:TCCTAAGCCA GTGCCAGAAG A 21 (2) SEQ ID NO:10:

(ⅰ) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:10:CTATTGGTCT CCTTAAACCT G 21 (2) SEQ ID NO:11:

(ⅰ) sequence signature:

(A) length: 23 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:11:CTCACAAACT AATGAAACCC TGC 23 (2) SEQ ID NO:12:

(ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:12:GAAAACAGCC CAAGGGACAG 20 (2) SEQ ID NO:13:

(ⅰ) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:13:TCCTAAGCCA GTGCCAGAAG A 21 (2) SEQ ID NO:14:

(ⅰ) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:14:CTATTGGTCT CCTTAAACCT G 21 (2) SEQ ID NO:15:

(ⅰ) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:15:CTCACAAACC TAATGAAACC CTGC 24 (2) SEQ ID NO:16:

(ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:16:GAAAACAGCC CAAGGGACAG 20 (2) SEQ ID NO:17:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:17:GCCCTGTGGG GCAAGGTGAA CGTGGAT 27 (2) SEQ ID NO:18:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:18:TACCCTTGGA CCCAGAGGTT CTTTGAG 27 (2) SEQ ID NO:19:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:19:GTTACTGCCC TGTGGGGCAA GGTGAAC 27 (2) SEQ ID NO:20:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:20:CACTTTGGCA AAGAATTCAC CCCACCA 27 (2) SEQ ID NO:21:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:21:GTGGTCTACC CTTGGACCCA GAGGTTC 27 (2) SEQ ID NO:22:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:22:CAGAGGTTCT TTGAGTCCTT TGGGGAT 27 (2) SEQ ID NO:23:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:23:AACCCTAAGG TGAAGGCTCA TGGCAAG 27 (2) SEQ ID NO:24:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:24:CTGCTGGTGG TCTACCCTTG GACCCAG 27 (2) SEQ ID NO:25:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:25:ACAGACACCA TGGTGCACCT GACTCCT 27 (2) SEQ ID NO:26:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:26:GTGCACCTGA CTCCTGAGGA GAAGTCY 27 (2) SEQ ID NO:27:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:27:CACCTGACTC CTGAGGAGAA GTCYGCN 27 (2) SEQ ID NO:28:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:28:ACTCCTGAGG AGAAGTCYGC NGTTACT 27 (2) SEQ ID NO:29:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:29:ACTCCTGAGG AGAAGTCYGC NGTTACT 27 (2) SEQ ID NO:30:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:30:GAGAAGTCTG CCGTTACTGC CCTGTGG 27 (2) SEQ ID NO:31:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:31:GCCGTTACTG CCCTGTGGGG CAAGGTGAAC 30 (2) SEQ ID NO:32:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:32:ACTGCCCTGT GGGGCAAGGT GAACGTG 27 (2) SEQ ID NO:33:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:33:GTTGGTGGTG AGGCCCTGGG CAGGCTGCTG 30 (2) SEQ ID NO:34:

(ⅱ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:34:CTGCCTGGTGG TCTACCCTTG GACCCAG 27 (2) SEQ ID NO:35:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:35:CTGGTGGTCT ACCCTTGGAC CCAGAGGTTC 30 (2) SEQ ID NO:36:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:36:GTGGTCTACC CTTGGACCCA GAGGTTCTTT 30 (2) SEQ ID NO:37:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:37:TGGACCCAGA GGTTCTTTGA GTCCTTTGGG 30 (2) SEQ ID NO:38:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:38:AGGTTCTTTG AGTCCTTTGG GGATCTG 27 (2) SEQ ID NO:39:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:39:GAGTCCTTTG GGGATCTGTC CACTCCT 27 (2) SEQ ID NO:40:

(ⅱ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:40:GTGAAGGCTC ATGGCAAGAA AGTGCTC 27 (2) SEQ ID NO:41:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:41:GTGCTCGGTG CCTTTAGTGA TGGCCTG 27 (2) SEQ ID NO:42:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:42:GTGCTCGGTG CCTTTAGTGA TGGCCTGGCT 30 (2) SEQ ID NO:43:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:43:AGTGATGGCC TGGCTCACCT GGACAAC 27 (2) SEQ ID NO:44:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:44:CTGGACAACC TCAAGGGCAC CTTTGCCACA 30 (2) SEQ ID NO:45:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: unknown (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence description: the information of SEQ ID NO:45:GAGCTGCACT GTGACAAGCT GCACGTG 27 (2) SEQ ID NO:46:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:46:AACTTCAGGC TCCTGGGCAA CGTGCTGGTC 30 (2) SEQ ID NO:47:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:47:CTCCTGGGCA ACGTGCTGGT CTGTGTG 27 (2) SEQ ID NO:48:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:48:TTCACCCCAC CAGTGCAGGC NGCCTAT 27 (2) SEQ ID NO:49:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:49CAAACAGACA CCATGGTGCA CCTGACT 27 (2) SEQ ID NO:50:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:50:CAAACAGACA CCATGGTGCA CCTGACT 27 (2) SEQ ID NO:51:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:51:GAGGCCCTGG GCAGGTTGGT ATCAAGG 27 (2) SEQ ID NO:52:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ IDNO:52:GAGGCCCTGG GCAGGTTGGT ATCAAGG 27 (2) SEQ ID NO:53:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:53:GAGAACTTCA GGGTGAGTCT ATGGGAC 27 (2) SEQ ID NO:54:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:54:GCCCTGGGCA GGTTGGTATC AAGGTTA 27 (2) SEQ ID NO:55:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:55:GCCCTGGGCA GGTTGGTATC AAGGTTA 27 (2) SEQ ID NO:56:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:56:TTTCCCACCC TTAGGCTGCT GGTGGTC 27 (2) SEQ ID NO:57:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:57:ATCTTCCTCC CACAGCTCCT GGGCAAC 27 (2) SEQ ID NO:58:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:58:ATCTTCCTCC CACAGCTCCT GGGCAAC 27 (2) SEQ ID NO:59:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: unknown (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence description: the information of SEQ ID NO:59:TTTCCCACCC TTAGGCTGCT GGTGGTC 27 (2) SEQ ID NO:60:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:60:CTGGGCAGGT TGGTATCAAG GTTACAA 27 (2) SEQ ID NO:61:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:61:CTGGGCAGGT TGGTATCAAG GTTACAA 27 (2) SEQ ID NO:62:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:62:CTGGGCAGGT TGGTATCAAG GTTACAA 27 (2) SEQ ID NO:63:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:63:GAGGCCCTGG GCAGGTTGGT ATCAAGG 27 (2) SEQ ID NO:64:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:64:GAGGCCCTGG GCAGGTTGGT ATCAAGG 27 (2) SEQ ID NO:65:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:65:GGTGAGGCCC TGGGCAGGTT GGTATCA 27 (2) SEQ ID NO:66:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:66:ATCTTCCTCC CACAGCTCCT GGGCAACGTG 30 (2) SEQ ID NO:67:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:67:TATTTTCCCA CCCTTAGGCT GCTGGTGGTC 30 (2) SEQ ID NO:68:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:68:TCTCTCTGCC TATTGGTCTA TTTTCCC 27 (2) SEQ ID NO:69:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEO ID NO:69:TGCCTATTGG TCTATTTTCC CACCCTT 27 (2) SEQ ID NO:70:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:70:AAATTGTAAC TGATGTAAGA GGTTTCA 27 (2) SEQ ID NO:71:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:71:AGCAGCTACA ATCCAGCTAC CATTCTGCTT 30 (2) SEQ ID NO:72:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:72:TTTCTGGGTT AAGGCAATAG CAATATT 27 (2) SEQ ID NO:73:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:73:GAAGTTGGTG GTGAGGCCCT GGGCAGG 27 (2) SEQ ID NO:74:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:74:GTGGATGAAG TTGGTGGTGA GGCCCTG 27 (2) SEQ ID NO:75:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:75:GTTGGTGGTG AGGCCCTGGG CAGGCTG 27 (2) SEQ ID NO:76:

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:76:CTGTGGGGCA AGGTGAACGT GGATGAAGTT 30 (2) SEQ ID NO:77:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:77:AGCATCATCA CGAACCTCCT GTACCAT 27 (2) SEQ ID NO:78:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:78:CAGAAGAACG ACCTGGACGC AGTGGCA 27 (2) SEQ ID NO:79:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:79:CCTAAAAGCT TCGGCTACAG CTCGGTG 27 (2) SEQ ID NO:80:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:80:TGTCGTGGGC ATCAGGTGAG TGAGTCA 27 (2) SEQ ID NO:81:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:81:TGGGCCAGAT ACTTTGTGAA GTTCCTG 27 (2) SEQ ID NO:82:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:82:GGCTCCTGGG ATCGAGGGAT GCAGTAC 27 (2) SEQ ID NO:83:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:83:GGCTCCTGGG ATCGAGGGAT GCAGTAC 27 (2) SEQ ID NO:84:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:84:GGACCCAATT GGGTGCGTAA CTTTGTC 27 (2) SEQ ID NO:85:

(ⅱ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:85:GACATCACCA AGGACACGTT TTACAAA 27 (2) SEQ ID NO:86:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:86:GTCGTGCTAA ACCGCTCCTC TAAGGAT 27 (2) SEQ ID NO:87:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: the unknown

(ⅱ) molecule type: DNA (genome)

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:87:TACCTGTGGC GTCGCCAGTG ATGGAGC 27 (2) SEQ ID NO:88:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: Ch1

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:88:AGCGCCGCCT ACGCCCACTC GGCTGTTTTC AGCAGCGUGG GCGTAGGCGG CGCUGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:89:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: Ch2

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:89:AGCGCCGCCT ACACCCACTC GGCTGTTTTC AGCCGAGUGG GTGTAGGCGG CGCUGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:90:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: Ch3

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:90:GCGCGTTTTC GCGCAGCGCC GCCUACGCCC ACUCGGCUGT TTTCAGCCGA GTGGGCGTAG 60GCGGCGCT 68 (2) SEQ ID NO:91:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: Dh1

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:91:AGCGCCGCCT ACGCCCACTC GGCTGTTTTC AGCCGAGTGG GCGTAGGCGG CGCTGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:92:

(ⅰ) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:92:ACCCCCAGCG CCGCCTACAC CCACTCGGCT GACCGG 36 (2) SEQ ID NO:93:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: SC1

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:93:ACCTGACTCC TGAGGAGAAG TCTGCTTTTG CAGACUUCUC CTCAGGAGUC AGGUGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:94:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: SC2

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:94:ACCTGACTCC TGTGGAGAAG TCTGCTTTTG CAGACUUCUC CACAGGAGUC AGGUGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:95:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: SC3

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:95:ATCTGACTCC TGAGGAGAAG ACTGCTTTTG CAGUCUUCUC CTCAGGAGUC AGAUGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:96:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: SC4

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:96:ACCTGACTCC TGAGGAGAAG ACTGCTTTTG CAGUCUUCUC CTCAGGAGUC AGGUGCGCGT 60TTTCGCGC 68 (2) SEQ ID NO:97:

(ⅰ) sequence signature:

(A) length: 68 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: SC5

(B) position: 1...68

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:97:GCGCGTTTTC GCGCACCUGA CUCCTGTGGA GAAGUCUGCT TTTGCAGACT TCTCCACAGG 60AGTCAGGT 68 (2) SEQ ID NO:98:

(ⅰ) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅸ) feature:

(A) title/keyword: δ

(B) position: 1...25

(D) other information:

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:98:ATCTGACTCC TGAGGAGAAG ACTGC 25 (2) SEQ ID NO:99:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:99:CACCTGACTC CTGAGGAGAA GTCTGCC 27 (2) SEQ ID NO:100:

(ⅰ) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: DNA

(ⅹ ⅰ) sequence description: SEQ ID NO:100:GAAGTTGGTG GTGAGGCCCT GGGCAGG 27

Claims (20)

1. be up to the nucleic acid of one 3 ' end and one 5 ' end, it has one and does not match the section of base, the configuration of this section makes the described base of not matching that this nucleic acid has been divided into article one chain and second chain, described article one chain and second chain comprise first district and second district respectively, there are at least 15 Nucleotide in each district, wherein:

(a) Nucleotide in each Nucleotide in described first district and described second district is the Watson-Crick paired;

(b) first district comprises at least 8 ribonucleotides, with 2 '-deoxynucleotide be the Watson-Crick paired, these ribonucleotides form at least one ribonucleotide section, described section has 3 ribonucleotides at least.

(c) sequence in described first district or described second district is a Human genome wild-type allele fragments sequence.

2. the nucleic acid of claim 1, the Human genome that provides is not the ras gene.

3. the nucleic acid of claim 1, the sequence in wherein said first district or described second district is 5 '-AGC GCC GCC TAC GCC CAC TCG GCT GT-3 ' (the Nucleotide 1-26 of SEQ ID NO:88) or its fragment.

4. the nucleic acid of claim 2, the Human genome that provides are not the genes of coding alkaline phosphatase.

5. the nucleic acid of claim 1, wherein said first district is made up of the second ribonucleotide section of the first ribonucleotide section of 6 adjacent ribonucleotides, 3 ribonucleotides and the section that interleaves that disposes between the first and second ribonucleotide sections at least at least, and described insertion section comprises and has 32 '-deoxyribonucleotides at least.

6. the Nucleotide of claim 4, wherein said second district comprises about 20-100 2 '-deoxyribonucleotide.

7. the nucleic acid of claim 5, each forms the wherein said first and second ribonucleotide sections to about 13 ribonucleotides by about 6, and the described section that interleaves is made up of the individual 2 '-deoxyribonucleotide of about 4-20.

8. the nucleic acid of claim 5, each is made up of the wherein said first and second ribonucleotide sections about 8-12 ribonucleotide, and the described section that interleaves is made up of the individual 2 '-deoxyribonucleotide of about 4-15.

9. the nucleic acid of claim 5, the wherein said first and second ribonucleotide sections are made up of about 10 ribonucleotides respectively, and the described section that interleaves is made up of about 52 '-deoxyribonucleotides.

10. the nucleic acid of claim 3, wherein said Human genome is selected from beta-globin gene and glucose cerebrosidase gene.

11. the nucleic acid of claim 9, wherein said first or the sequence in second district be selected from the sequence of table I or be its fragment.

12. the nucleic acid of claim 9, wherein said first or the sequence in second district be 5 '-CACCTG ACT CCT GAG GAG AAG TCT GCC-3 ' (SEQ ID NO:99) or 5 '-GAA GTT GGT GGT GAG GCC CTG GGC AGG-3 ' (SEQ ID NO:100) or its fragment.

13. repair the method for the cell mutation of human subject, but comprise the curee of the disease that causes from the existence of suffering from CRV repair cell sudden change and take out cell, and nucleic acid is incorporated in the cell of taking-up, wherein said nucleic acid is up to one 3 ' end and one 5 ' end, and comprise one and do not match the section of base, the configuration of this section makes the described base of not matching that this nucleic acid has been divided into article one chain and second chain, described article one chain and second chain comprise first district and second district respectively, there are at least 15 Nucleotide in each district, wherein:

(a) Nucleotide in each Nucleotide in described first district and described second district is the Watson-Crick paired;

(b) described first district comprises at least 8 ribonucleotides, with 2 '-deoxynucleotide be the Watson-Crick paired, these ribonucleotides form at least one section, described section has 3 ribonucleotides at least; And

(c) sequence in described first district or described second district is a Human genome wild-type allele fragments sequence, and described fragment comprises CRV can repair sudden change.

14. the method for claim 12, the sequence in wherein said first district or described second district are 5 '-AGC GCC GCC TAC GCC CAC TCG GCT GT-3 ' (the Nucleotide 1-26 of SEQ ID NO:88) or its fragment.

15. the method for claim 12, it also comprises implants the intravital step of described curee again to the cell of repairing.

16. the method for claim 13, first district of wherein said nucleic acid is made up of the first ribonucleotide section, the second ribonucleotide section of 6 adjacent ribonucleotides and the section that interleaves that disposes between the described first and second ribonucleotide sections at least, described insertion section comprises at least 32 '-deoxyribonucleotide, and wherein said cell is a hemopoietic stem cell.

17. the method for claim 14, the wherein said section that interleaves comprises described CRV and can repair sudden change.

18. the method for claim 14, wherein said Human genome are selected from beta-globin gene and glucose cerebrosidase gene.

19. the method for claim 16, the sequence in wherein said first district or second district are selected from sequence or its fragment of table I.

20. the method for claim 16, the sequence in wherein said first district or second district are 5 '-CAC CTG ACT CCT GAG GAG AAG TCT GCC-3 ' (SEQ ID NO:99) or 5 '-GAA GTT GGT GGT GAG GCC CTG GGC AGG-3 ' (SEQ ID NO:100) or its fragment.

Images